The long-term objective of this project is to understand the pathogenesis of Lassa virus. A potential bioterrorism agent, Lassa virus causes hemorrhagic fever in humans and has been classified as a 'Category A Priority Pathogen' in the NIAID Biodefense and Emerging Diseases Research Opportunities Program. Worldwide, this virus causes an estimated 100,000 to 300,000 infections and approximately 5,000 deaths annually. Yet, no satisfactory antiviral compounds or protective vaccines are available, largely because of the lack of available systems that permit dissection of the viral life cycle at the molecular level. Thus, an immediate goal of this proposal is to establish a system for the generation of Lassa virus entirely from cloned cDNA, building on the applicant's experience with the artificial generation influenza and Ebola viruses. Lassa virus generated from cloned cDNA will then be used to assess the biological significance of the viral glycoprotein cleavage sequence, which is unusual in that the glycoprotein is cleaved after a nonbasic amino acid, in contrast to most other viruses whose surface glycoproteins are cleaved after a basic residue. Additional objectives are: (i) development of a minireplicon system in which a virus-like RNA encoding a reporter protein is replicated by the nucleoprotein and polymerase protein. This system would allow us to address important questions about Lassa viral replication and transcription; and (ii) development of a system in which expression of all viral proteins and a virus-like RNA yields virus-like particles, allowing examination of virus assembly, budding, attachment, and internalization processes at the molecular level. The system for the de novo synthesis of Lassa virus proposed here is timely, as it will permit identification of determinants of Lassa virus pathogenicity, a necessary first step in the development of antiviral compounds and protective vaccines.